The only example of an adenosylcobalamin (AdoCbl)-dependent isomerase in mammals is the mitochondrial enzyme, methylmalonyI-CoA mutase, which catalyzes the rearrangement of methylmalonyI-CoA to succinyI-CoA. Dysfunction of methylmalonyI-CoA mutase leads to methylmalonic aciduria, which causes aberrations in organic acid metabolism, and in severe cases, can be fatal. This proposal focuses on elucidating the reaction mechanism of methylmalonyI-CoA mutase, and characterizing the biochemical penalties associated with specific missense mutations that are pathogenic. The role of the cofactor, AdoCbl, in this reaction is to serve as a latent radical reservoir which is deployed during catalysis to accomplish a chemically challenging carbon skeleton rearrangement reaction. The enzyme orchestrates an ca. trillion fold rate enhancement of the homolytic cleavage of the cobalt-carbon (Co-C) bond in the presence of substrate, and its mechanism has been the subject of enduring debate. We propose to (i) elucidate the mechanism by which the Co-C bond is labilized by testing the hypothesis that Y89, an active site residue, is used as a molecular wedge to pry apart the Co-C bond following binding of substrate. We will examine the effect of substitutions at Y89 on kinetic coupling and tunneling and use spectroscopic methods to probe the electronic environment of the corrin in wild type and Y89 variants in the presence of substrate analogs. (ii) We will elucidate the roles of the "aromatic corridor" residues in the active site on stereochemical control of the rearrangement, and estimate radical geometries and interradical distances by pulsed EPR methods, and spectral simulations. Substrate analogs that are designed to stabilize the radical intermediates will be designed. (iii) We will characterize the biochemical deficits associated with a select group of missense mutations identified in methylmalonic aciduria patients and functional heterodimers carrying either an N-terminal R93H mutation or a C-terminal mutation (in the AdoCbl-binding domain) in each subunit to determine the basis of the observed complementation. These studies on the wild type and pathogenic variants of methylmalonyI-CoA mutase will make inroads into our understanding of a novel and clinically important enzyme. [unreadable] [unreadable]